Structural and functional criteria reveal a new nuclear import sequence on the 5-lipoxygenase protein

Leukotrienes are lipid mediators with important roles in immunity. The enzyme 5-lipoxygenase initiates leukotriene synthesis; nuclear import of 5-lipoxygenase modulates leukotriene synthetic capacity. In this study, we used structural and functional criteria to identify potential nuclear import sequences. Specifically, we sought basic residues that 1) were common to different 5-lipoxygenases but not shared with other lipoxygenases, 2) were found on random coil/loop structures, and 3) could be replaced without eliminating catalytic activity. Application of these criteria to the putative bipartite nuclear import sequence of 5-lipoxygenase revealed that this region formed an alpha-helix rather than a random coil, that the critical residue arginine 651 serves a structural role, and that mutation of this residue eliminated catalytic activity. A previously unrecognized region corresponding to residues 518-530 on human 5-lipoxygenase was found to be unique to 5-lipoxygenase and on a random coil. This region alone was sufficient to drive import of green fluorescent protein to the same degree as complete 5-lipoxygenase. Replacement of basic residues in this region of the complete protein was capable of eliminating nuclear import without abolishing catalytic activity. Surprisingly, two subpopulations of cells expressing 5-lipoxygenase with this mutated region could be discerned: those with strongly impaired import and those with normal import. Taken together, these results show that the previously identified region with a bipartite motif is not a functional import sequence, whereas the newly identified basic region constitutes a true nuclear import sequence. Moreover, we suggest that another sequence that can mediate nuclear import of 5-lipoxygenase remains to be identified.     

Expression in E. coli and purification of Thermus thermophilus translation initiation factors IF1 and IF3

The initiation of protein translation in bacteria requires in addition to mRNA, fMet-tRNA, and ribosomal subunits three protein factors, the initiation factor 1 (IF1), initiation factor 2 (IF2), and initiation factor 3 (IF3). The genes coding for IF1 and IF3 from Thermus thermophilus have been identified and cloned into pET expression vector and were expressed as soluble proteins in Escherichia coli. IF1 was purified by a DEAE-cellulose chromatography, followed by heat denaturation, chromatography on Hydroxylapatit, and gel permeation chromatography using Sephacryl 200HR. For the purification of IF3, a heat denaturation step is followed by anion-exchange chromatography on Q-Sepharose FF and gel permeation chromatography on Sephacryl 200HR. Using these procedures we obtained chromatographically pure and biologically active preparations of both T. thermophilus IF1 and IF3.     

Leukotriene synthesis by epithelial cells

Leukotrienes (LTs) are intercellular signaling molecules that evoke a variety of responses. They are best known as potent promoters of inflammation. Normally, LTs are produced primarily by leukocytes. As a result, current models regarding the production of LTs in the context of disease focus on the leukocytes as the site of production. Structural cells, including epithelial cells, are typically relegated to supportive roles. It is recognized that epithelial cells normally contain all the components necessary for LT synthesis except the enzyme 5-lipoxygenase (5-LO). There is accumulating evidence that some populations of epithelial cells normally express low levels of 5-LO and can synthesize LTs autonomously. Moreover, certain factors, including bacterial and viral infection, can promote the expression of 5-LO in airway, gastrointestinal and skin epithelial cells. The appearance of active 5-LO enzyme in epithelial cells at these sites may contribute to diseases like cancer, colitis and psoriasis. This paper reviews the state of our knowledge regarding the expression of 5-LO in epithelial cells, the factors that modify that expression, and the implications regarding pathogenesis.     

Kupffer cell-initiated remote hepatic injury following bilateral hindlimb ischemia is complement dependent

Intravital fluorescence microscopy was applied to the livers of male Wistar rats to test the hypothesis that complement mobilization stimulates Kupffer cells and subsequently initiates hepatic injury after hindlimb ischemia/reperfusion (I/R). Following 3 h of limb reperfusion, hepatocellular viability (serum levels of alanine transaminase and cell death via propidium iodide labeling) decreased significantly from levels in sham-operated animals. Inhibition of complement mobilization with soluble complement receptor type 1 (20 mg/kg body wt) and interruption of Kupffer cell function with GdCl(3) (1 mg/100g body wt) resulted in significant hepatocellular protection. Although the effects of hindlimb I/R on hepatic microvascular perfusion were manifest as increased heterogeneity, both complement inhibition and suppression of Kupffer cell function resulted in marked improvements. No additional hepatocellular protection and microvascular improvements were provided by combining the interventions. Furthermore, inhibition of complement mobilization significantly depressed Kupffer cell phagocytosis by 42% following limb reperfusion. These results suggest that the stimulation of Kupffer cells via complement mobilization is necessary but is not the only factor contributing to the early pathogenesis of hepatic injury following hindlimb I/R.     

Mutational analysis of the C-terminus in ion transport peptide (ITP) expressed in Drosophila Kc1 cells

Ion transport peptide (ITP) stimulates Cl(-) transport (measured as short-circuit current, I(sc)) and fluid reabsorption in Schistocerca gregaria ilea. We report that Drosophila Kc1 cells transfected with preproITP cDNA secrete a peptide (KcITP(75)) that, while cleaved correctly at the N-terminus, had reduced (10-fold) stimulatory activity on ileal I(sc) compared to both native ITP (ScgITP) and synthetic ITP (synITP). We provide evidence that the reduced activity of KcITP(75) is due to incomplete processing of the C-terminal sequence LGKK (KcITP(75)) to L-amide. In support of this, in vitro amidation of glycine extended ITP (i.e., KcITP(73) ending in LG) but not KcITP(75) (ending in LGKK) significantly increased specific activity in the bioassay. Further evidence for C-terminus involvement includes complete loss of stimulation by truncated mutants (e.g., KcITP(71) which lacks LGKK) and a mutant in which alanine is substituted for the terminal glycine in KcITP(73). Moreover a natural homologue (KcITP-L, which differs only in the C-terminal sequence) expressed by Kc1 cells does not stimulate ileal I(sc). Rather KcITP-L acts as a weak ITP antagonist, as does the truncated mutant KcITP(71). KcITP(70) has no antagonistic effect. A short synthetic peptide fragment of the C-terminus (VEIL-amide) does not stimulate ileal I(sc), indicating that other regions of ITP are also essential to biological activity. Arch.     

The neuronal progenitor cells of the forebrain subventricular zone: intrinsic properties in vitro and following transplantation

During the development of the central nervous system, progenitor cells, located within distinct germinal zones, produce presumptive neurons that migrate to their destinations and differentiate. Recent studies have demonstrated that a discrete region of the anterior part of the postnatal subventricular zone (SVZa) comprises neuronal progenitor cells whose progeny are fated to become the interneurons of the olfactory bulb. The SVZa is of particular interest because it is one of few germinal zones to persist postnatally and may be the only postnatal germinal zone to give rise exclusively to neurons. To the extent that the SVZa is unique among proliferative zones, the SVZa progeny are unique among neurons. First, unlike most cortical neurons, the SVZa-derived cells do not rely on radial glia-assisted migration when traveling to their target region. Second, the SVZa progeny continue to proliferate as they migrate to their target region. And third, the SVZa progeny express early neuron-specific antigens prior to their final division and, therefore, prior to reaching their destination where they will terminally differentiate. To better understand the capacity of the SVZa progeny to concurrently proliferate, migrate, and differentiate, we studied the cells in vitro and following transplantation into the neonatal SVZa and adult striatum. In each setting, we found that the SVZa cells continue both to proliferate and to differentiate into neurons. In addition, after homotopic and heterotopic transplantation, we found that the SVZa cells maintain their ability to migrate. These results suggest that the unique features of the SVZa progeny are specified intrinsically rather than by their extrinsic environment.     

Inhibition of photosystem II (PSII) electron transport as a convenient endpoint to assess stress of the herbicide linuron on freshwater plants

Effects of the herbicide linuron on photosynthesis of the freshwater macrophytes Elodea nuttallii (Planchon) St. John, Myriophyllum spicatum L., Potamogeton crispus L., Ranunculus circinatus Sibth., Ceratophyllum demersum L. and Chara globularis (Thuill.), and of the alga Scenedesmus acutus Meyen, were assessed by measuring the efficiency of photosystem II electron flow using chlorophyll fluorescence. In a series of single-species laboratory tests several plant species were exposed to linuron at concentrations ranging from 0 to 1000 μg l−1. It was found that the primary effect of linuron, inhibition of photosystem II electron flow, occurred with a half-lifetime of about 0.1 to 1.9 h after addition of linuron to the growth medium. The direct effect of the herbicide on photosynthesis appeared to be reversible. Complete recovery from the inhibition occurred with a half-lifetime of 0.5 to 1.8 h after transfer of linuron treated plants to linuron free medium. The EC50,24h of the inhibition of photosystem II electron transport by linuron was about 9–13 μg l−1 for most of the macrophytes tested. For S. acutus the EC50,72h for inhibition of photosystem II electron flow was about 17 μg l−1 for the free suspension, and 22 μg l−1 for cells encapsulated in alginate beads. In a long-term indoor microcosm experiment, the photosystem II electron flow of the macrophytes E. nuttallii, C. demersum and the alga Spirogyra sp. was determined during 4 weeks of chronic exposure to linuron. The EC50,4weeks for the long-term exposure was 8.3, 8.7 and 25.1 μg l−1 for E. nuttallii, C. demersum and Spirogyra, respectively. These results are very similar to those calculated for the acute effects. The relative biomass increase of E. nuttallii in the microcosms was determined during 3 weeks of chronic exposure and was related to the efficiency of photosystem II electron transport as assessed in the different treatments. It is concluded that effects of the photosynthesis inhibiting herbicide on aquatic macrophytes, algae and algae encapsulated in alginate beads can be conveniently evaluated by measuring photosystem II electron transport by means of chlorophyll fluorescence. This method can be used as a rapid and non-destructive technique in aquatic ecological research. This revised version was published online in August 2006 with corrections to the Cover Date.     

Ecotoxicological threshold levels of a mixture of herbicides (atrazine, diuron and metolachlor) in freshwater microcosms

Twelve indoor, plankton-dominated, freshwater microcosms (600 l) were used to study the effect of a mixture of herbicides on structural and functional aspects of these ecosystems. The EC50, 72 h values of the most susceptible standard test alga Selenastrum capricornutum (EC50, atrazine=54 μg l−1, EC50, diuron=15 μg l−1, EC50, metolachlor=56 μg l−1) were used as a starting point for the dosage applied in the microcosms (dosages: 0, 0.01, 0.03, 0.1, 0.3, 1× EC50). The microcosms were exposed to chronic levels for 28 days and subsequently monitored for 4 more weeks. The following effects were observed: (1) direct effects became apparent from an initial drop in photosynthesis efficiency, pH and oxygen concentration and a decrease in the abundance of several phytoplankton taxa at the 0.3 × EC50 treatment level and higher. (2) Fourteen days post application an increase in the abundance of several phytoplankton taxa (Chlamydomonas sp. and Stephanodiscus/Cyclotella) was observed; oxygen concentrations recovered while alkalinity, conductivity and total inorganic nitrogen were elevated. (3) Effects on fauna were minor. Daphnia galeata showed a decreasing trend and the cyclopoid copepods an increasing trend at the end of the experiment. Multivariate statistical analyses demonstrated no effects of any treatment level on the zooplankton community. Effects were reported for the phytoplankton community at dose levels of 0.3 × EC50 and higher. On species level the most sensitive taxon was Chlorophyceae coccales. For this taxon a NOEC at the dose level of 0.01 × EC50 was calculated. This effect however was relatively small in magnitude and merely based on an increase in numbers in the control and lowest treated microcosms rather than a decrease in numbers in all other treatments. The standards based on algal toxicity data, as adopted by the Uniform Principles, consist of a safety factors of 0.1 to be multiplied with the EC50. The NOEC of coccales was lower than 0.1 × EC50. All other observed variables in this aquatic ecosystem were sufficiently protected against the mixture of herbicides by the safety factor as proposed in the Uniform Principles. This revised version was published online in August 2006 with corrections to the Cover Date.